1. Technical Field
The present invention relates to: a composition used in preparing paraffin block by collecting biological tissues, biological fluids, or single cells and preparing them to a mass, assemblage and formation for microscopic observation, biomonitoring, test, disease diagnosis, prediction, verification, detection, confirmation or selection, and disease monitoring (hereinafter, ‘a composition for aggregating biological sample’); a method for preparing paraffin block using the composition for aggregating biological sample for study of the sample; and a method for microscopic observation of the biological sample aggregated by the composition for aggregating biological sample and fixed in the paraffin block to distinguish between tissues with and without disease, and cells with and without disease.
In addition, the present invention relates to a method for preparing cell block for cellular microarray including a process to aggregate biological samples such as tissue cells and cultured cells using the composition for aggregating biological sample and a cell block for cellular microarray prepared by the same.
Using the composition for aggregating biological sample of the present invention, it is possible to improve accuracy of histological and cytological diagnosis for extremely small amount and fluid sample and improve the cell block technique to higher quality cell block technique in order to allow conservative diagnostic approaches by maintaining antigenicity of the paraffin block prepared from the sample.
2. Description of the Related Art
Histological or cytological examination not only takes a very important position in laboratory medicine but also plays an important role in making a final decision of clinical disease diagnosis or studying disease. Pathological examination is an examination that isolates or aspirates human tissue suffering a disease to identify the name of disease such as cancer through microscopic observation. The histological or cytological examination is based on observing and judging morphological changes of cells or tissues different from other laboratory methods. Nowadays, a lot of efforts have been given to obtain these data and for this, various special methods have been used according to a trend wanting to get objective data.
Especially recently, histochemical examination, immunohistochemistry, special staining, and molecular pathological examination have appeared to increase accuracy of objective diagnosis. For accurate diagnosis of diseases and study on proper therapies for them, it is important to observe morphological findings of biological sample collected from lesions. In order to observe these morphological changes, researchers use hematoxylin-eosin staining specimen basically. However, because various diseases has been increasingly segmented, new diagnostic techniques have been being introduced increasingly, and a lot of tumor markers helpful to tumor diagnosis and more prognostic factors able to predict prognosis have got known, it is difficult to obtain comprehensive information with conventional hematoxylin-eosin staining specimen. Examination methods for complementing these problems include immunohistochemical staining method and molecular pathologic evaluation. Among these, the immunohistochemical staining is being used efficiently for identifying the origin of undifferentiated cell, determining existence of enzyme, hormone, tumor marker, and prognostic factor, discriminating between carcinoma and sarcoma, discriminating between benign tumor and malignant tumor, and presuming primary lesion of metastatic cancer. It is highly sensitive and specific examination method, which has been used as an essential examination method in diagnostic pathology and morphological study for the last 20˜30 years.
Diagnostic cytology is a field of pathology to determine existence or contents of a lesion in aspects of morphology based on cytological findings, which refers to a clinical laboratory medicine to interpret microscopically cells exfoliated naturally from various regions of human body by several clinical manipulations or cells detached from tissue surface or inside with an instrument. The diagnostic cytology includes: i) spontaneous exfoliative diagnostic cytology that smears cells exfoliated spontaneously by normal or pathological causes on a slide and then observes them with a microscope after fixation and staining; ii) aspiration biopsy diagnostic cytology that aspirates cells by injecting a fine needle attached syringe into the lesion, collects cells by compulsion, smears them on a slide, and observes them with a microscope after fixation and staining; iii) touch print diagnostic cytology that makes a slide contact directly to a lesion visible with naked eyes, smears cells on it, and then observes them with a microscope after fixation and staining; iv) cell smear examination that smears target cells collected on a slide thinly and then observes them with a microscope; v) cell block technique that prepares paraffin embedded cell block by infiltrating paraffin into cellular sediments collected from liquid specimen; and vi) sputum examination. Among the diagnostic cytological methods, the cell block technique retains superior merits to other methods and keeps remarkable diagnostic values in the points that is capable of special staining to identify other cell products, bacteria and fungi, preparing fluid specimen, observing diagnostic evidences which is impossible to observe them in other methods.
In application of the cell block method, the liquid sample is collected from various regions of human body and concretely, the effluents from respiratory tract, gastrointestinal system, and urinary system is made to the cell block by fixing in a fixative at first and then prepared to the paraffin block. The conventional methods to prepare cell block that have been used include: i) a fixed sediment method using sedimentation with centrifuging and formalin; ii) an egg albumin method using a principle that the egg albumin is coagulated in ethanol; iii) an agar method; iv) a plasma thrombin clot method; v) collodion bag method; and vi) Millpore filter method.
However, these traditional methods actually cannot get satisfactory results because of several problems including difficulty in slide interpretation from background staining, contraction of sample supporting agar in the method using agar gel, time required for preparing paraffin block and high cost.
Besides, while the paraffin block method has been used for the purpose of diagnosis, treatment and study through understanding of shapes of tissue and cells, special staining and immunohistochemical staining from it, it has been difficult or impossible to prepare the paraffin block when the quantity of collected cells was extremely little or the sample is extremely small. In addition, when preparing a paraffin block from tissue or cellular suspension with centrifuging, there have been some problems also, including that because of small size or quantity of tissue, the tissue or cells were dispersed, discrimination with paraffin was difficult, and paraffin embedding with a pincette was impossible.
Korea Public Patent Notification No. 2009-97068 describes a composition of embedding resin for histological analysis of scaffold for tissue engineering and a slide preparing method by the same and concretely, describes a method including following steps: performing melt blending of ethylene vinyl acetate copolymer containing paraffin and vinyl acetate as 1-80% at 40-100° C. to 2-50%; and then making the paraffin and ethylene derived copolymer blended solution infiltrated by adding a sample to the melt.
In addition, Japan Public Patent Notification No. 2007-046988 describes a method for preparing embedded block for biological tissue specimen and information label for the embedded block, Korea Public Patent Notification No. 2005-46826 describes a method of using laser capture microdissection (LCM) extracted in pure RNA that obtained from paraffin block through tissue specific fixing agent, and WO 08/038813 describes a method to perform deparaffinization process from paraffin embedded specimen and analyze it.
Besides, the cellular microarray means an experimental system to obtain multiple information on genetic and protein alteration in different cell and their interaction by analyzing antibodies, proteins, lipids, or staining substances using cell block specimen where different kinds or tissues or cultured cells were arrayed on a solid scaffold Chen D S et al, Curr Opin Chem Biol (2006), 10:28-34]. Generally, as the specimen for cellular microarray is prepared by making block and sectioning after microarray of cell pellets obtained from cell bock prepared in the in a fixed case, the results depends on aggregation and embedding process in the cell block preparing process largely.
As shown in several studies on preparation of cell block relating to cellular microarray including Waterworth A et al, In Vitro Cell DevBiolAnim (2005), 41(7):185-187 and US Patent Publication No. 2010/0323907, 2006/0160169, and 2003/0157523, there were only cellular microarray techniques by paraffin infiltration or frozen section after fixation with formalin or agar in general methods, studies on methods for preparing cell block to solve the problems were actually insufficient.
Therefore, it is required constantly to develop a method for preparing cell block for cellular microarray able to satisfactory multiple diagnostic information for cells with small quantity or size.